title: Oxidative stress precedes skeletal muscle mitochondrial dysfunction during experimental aortic cross-clamping but is not associated with early lung, heart, brain, liver, or kidney mitochondrial impairment.
abstract: OBJECTIVE
  Lower limb ischemia-reperfusion results in skeletal muscle mitochondrial alterations, production of reactive oxygen species (ROS), and remote organ impairments that are largely involved in patient prognosis. However, whether ischemia without reperfusion increases ROS production and precedes mitochondrial alteration and whether mitochondrial dysfunction occurs early in remote organs is unknown. This study determined muscle mitochondrial function and ROS production after ischemia alone, or followed by two periods of reperfusion, and investigated heart, lung, liver, kidney, and brain mitochondrial functions after lower limb ischemia-reperfusion.
  
  
  METHODS
  Wistar rats were randomized into four groups: sham (aortic exposure but no ischemia, n = 9), I3 (ischemia alone induced by aortic cross-clamping for 3 hours, n = 9), I3R10' and I3R2 (aortic cross-clamping, followed by reperfusion for 10 minutes [n = 8] or 2 hours [n = 9]). Blood lactate, alanine aminotransferase, aspartate aminotransferase, and creatinine were measured. Mitochondrial respiratory chain complexes I, II, III, and IV activities and mitochondrial coupling (acceptor control ratio) were analyzed using a Clark oxygen electrode in skeletal muscle, lung, heart, brain, liver, and kidney. ROS production was determined using dihydroethidium staining in muscle, heart, liver, and kidney. Inflammation was also investigated in remote organs (heart, liver, and kidney) using monocyte-macrophage-2 antibody staining.
  
  
  RESULTS
  Lactate level increased after ischemia in all groups. In muscle, ROS increased significantly after ischemia alone (+324% ± 66%; P = .038), normalized after 10 minutes of reperfusion, and increased again at 2 hours of reperfusion (+349.2 ± 67%; P = .024). Interestingly, mitochondrial function was unaffected by ischemia alone or followed by 10 minutes of reperfusion, but maximal mitochondrial oxidative capacity (6.10 ± 0.51 vs. 4.24 ± 0.36 μmol/min/g, -30%; P < .05) and mitochondrial coupling decreased after 2 hours of reperfusion (1.93 ± 0.17 vs. 1.33 ± 0.07, -45%; P < .01), in sham and I3R2 rats, respectively. Despite increased serum aspartate aminotransferase (×13; P < .0001), alanine aminotransferase (×6; P = .0019), and creatinine (×3; P = .0004), remote organs did not show mitochondrial alteration, inflammation, or ROS production enhancement after 2 hours of reperfusion.
  
  
  CONCLUSIONS
  Oxidative stress precedes skeletal muscle mitochondrial dysfunction during lower limb ischemia. Such a kinetic explains the efficacy of ischemic preconditioning and supports that therapy should be conducted even during ongoing ischemia, suggesting that ischemic preconditioning might be a successful approach.
authors.affiliation: Équipe d'accueil 3072, Institut de Physiologie, Faculté de Médecine, Fédération de Médecine Translationelle, Université de Strasbourg, Strasbourg, France; Service de Réanimation Médicale, Pôle d'Urgences, Réanimations Médicales, Centre Anti-Poison, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.
authors.forename: Max
authors.initials: M
authors.lastname: Guillot
authors.affiliation: Équipe d'accueil 3072, Institut de Physiologie, Faculté de Médecine, Fédération de Médecine Translationelle, Université de Strasbourg, Strasbourg, France; Service de Physiologie et d'Explorations Fonctionnelles, Pôle de Pathologies Thoraciques, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.
authors.forename: Anne-Laure
authors.initials: AL
authors.lastname: Charles
authors.affiliation: Équipe d'accueil 3072, Institut de Physiologie, Faculté de Médecine, Fédération de Médecine Translationelle, Université de Strasbourg, Strasbourg, France; Pôle d'Anesthésie, Réanimation Chirurgicale, SAMU, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.
authors.forename: Thien Nga
authors.initials: TN
authors.lastname: Chamaraux-Tran
authors.affiliation: Laboratory 410, Clinical Pharmacology and Toxicology, Department of Biomedicine, University Hospital Basel, Basel, Switzerland.
authors.forename: Jamal
authors.initials: J
authors.lastname: Bouitbir
authors.affiliation: Équipe d'accueil 3072, Institut de Physiologie, Faculté de Médecine, Fédération de Médecine Translationelle, Université de Strasbourg, Strasbourg, France; Service de Physiologie et d'Explorations Fonctionnelles, Pôle de Pathologies Thoraciques, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Meyer
authors.affiliation: Équipe d'accueil 3072, Institut de Physiologie, Faculté de Médecine, Fédération de Médecine Translationelle, Université de Strasbourg, Strasbourg, France; Service de Physiologie et d'Explorations Fonctionnelles, Pôle de Pathologies Thoraciques, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.
authors.forename: Joffrey
authors.initials: J
authors.lastname: Zoll
authors.affiliation: Service de Réanimation Médicale, Pôle d'Urgences, Réanimations Médicales, Centre Anti-Poison, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.
authors.forename: Francis
authors.initials: F
authors.lastname: Schneider
authors.affiliation: Équipe d'accueil 3072, Institut de Physiologie, Faculté de Médecine, Fédération de Médecine Translationelle, Université de Strasbourg, Strasbourg, France; Service de Physiologie et d'Explorations Fonctionnelles, Pôle de Pathologies Thoraciques, Hôpitaux Universitaires de Strasbourg, Strasbourg, France. Electronic address: bernard.geny@chru-strasbourg.fr.
authors.forename: Bernard
authors.initials: B
authors.lastname: Geny
mesh_terms: D000818:Animals; D001012:Aorta, Abdominal / Q000601:surgery; D004195:Disease Models, Animal; D007150:Immunohistochemistry; D019194:Ischemic Preconditioning; D007668:Kidney / Q000378:metabolism* / Q000473:pathology; D008099:Liver / Q000378:metabolism* / Q000473:pathology; D035002:Lower Extremity / Q000098:blood supply; D008168:Lung / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D028361:Mitochondrial Diseases / Q000209:etiology / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism* / Q000473:pathology
chemical_list: D017382:Reactive Oxygen Species